Chlorpyrifos (CPF), a phosphorothioate insecticide, is metabolically activated by cytochrome P450 enzymes (CYPs) to a potent anticholinergic metabolite, chlorpyrifos-oxon, and inactivated by several detoxification pathways. Therefore, the overall balance between metabolic generation and elimination of the toxic product is a major determinant of CPF toxicity. Because CYPs are critical in the activation of CPF, this proposal is designed to test hypothesis 1, that CPF is differentially activated by multiple forms of CYPs and detoxified largely by microsomal carboxylesterases and A-esterases in male and female rats. The in vivo model system presented here is designed to identify which CYP enzymes are responsible for CPF activation by using their specific substrates and inducers as useful probes. The magnitude of inhibition of the CYPs, esterases, and plasma and brain cholinesterases will be used as an indicator of CPF activation and toxicity. The in vitro model system will be used to test hypothesis 2, that the differences in activation and toxicity of CPF between males and females may be due to toxicokinetic influences. Hypothesis 2 will test whether the differences in CPF toxicity in males and females are due to the differential capacity of CYP enzymes to activate CPF, and whether their selective chemical inhibitors and specific inhibitory monoclonal antibodies (MAbs) can be used to definitively identify the CPF-activating CYP isoforms. Hypothesis 3, that there may be distinct differences in the activation and toxicokinetics of CPF by hepatic CYP enzymes between rats and humans, will be tested by using male and female human liver microsomes as well as expressed human P450s. These experiments are expected to identify and characterize the most important CYPs involved in the conversion of CPF to its oxon in rats and humans. In summary, the proposed studies will define the role and determine the capacity of hepatic P450 enzymes to biotransform CPF in males and females, which will enable us to make a more accurate assessment of possible differences in rat and human susceptibility to this and other organophosphate insecticides.